From 2014.igem.org
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| = Agar Chips = | | = Agar Chips = |
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| = Transformation = | | = Transformation = |
Revision as of 23:24, 17 September 2014
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Media
LB medium
- weight components
- 5 g/L NaCl
- 10 g/L tryptone
- 5 g/L yeast extract
- (15 g/L agar for plates)
- fill up to 1 L with deionized water
- autoclave
- autoclaving tape, caps slightly unscrewed
- base of the pot has to be covered with deionized water
- close lid
- heat level 3 until the pressure valve opens
- reduce heat level to 1.5
- set timer to 20 minutes
- turn heater off
- wait until the pressure valve retracts (30-45 minutes)
- open, close caps & shake
- for plates, wait until you can touch the bottle (<60 °C, clean bench!)
- add antibiotics and shake (gloves!)
Hartmans minimal medium (HM)
Component | supplement for 1x HM [g/L] | final 1x concentration
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Casamino acids | 2 | 2 g/L
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Thiamine hydrochloride | 0.3 | 300 mg/L
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MgSO4/MgSO4*7H2O | 0.242 / 0,494 | 2 mmol/L
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CaCl2 | 0.011 | 0.1 mmol/L
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Agar Chips
Transformation
Heat Shock
- thaw cells on ice
- add 1 µl of plasmid DNA
- incubate on ice for 30'
- heat shock at 42 °C for 60
- incubate on ice for 5'
- add 200 µl of SOC media
- incubate at 37 °C for 2 h
- plate 20 and 200 µl on plates with the appropiate antibiotic
Electroporation
PCR
Colony PCR
QuikChange
Clonings
Restriction Digest
Ligation
Gibson Assembly
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